Sekhar Pagadala Nataraj, Arha Manish, Reddy P S, Kumar Ranadheer, Sirisha V L, Prashant S, Janardhan Reddy K, Khan Bashir, Rawal S K, Kavi Kishor P B
Department of Genetics, Osmania University, Hyderabad 500007, India.
J Mol Model. 2009 Feb;15(2):203-21. doi: 10.1007/s00894-008-0395-8. Epub 2008 Dec 2.
Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) is an important enzyme that participates in lignin biosynthesis especially in the formation of cell wall ferulic esters of plants. It plays a pivotal role in the methylation of the 3-hydroxyl group of caffeoyl CoA. Two cDNA clones that code CCoAOMT were isolated earlier from subabul and in the present study; 3D models of CCoAOMT1 and CCoAOMT2 enzymes were built using the MODELLER7v7 software to find out the substrate binding sites. These two proteins differed only in two amino acids and may have little or no functional redundancy. Refined models of the proteins were obtained after energy minimization and molecular dynamics in a solvated water layer. The models were further assessed by PROCHECK, WHATCHECK, Verify_3D and ERRAT programs and the results indicated that these models are reliable for further active site and docking analysis. The refined models showed that the two proteins have 9 and 10 alpha-helices, 6 and 7 beta-sheets respectively. The models were used for docking the substrates CoA, SAM, SAH, caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapyl CoA which showed that CoA and caffeoyl CoA are binding with high affinity with the enzymes in the presence and absence of SAM. It appears therefore that caffeoyl CoA is the substrate for both the isoenzymes. The results also indicated that CoA and caffeoyl CoA are binding with higher affinity to CCoAOMT2 than CCoAOMT1. Therefore, CCoAOMT2 conformation is thought to be the active form that exists in subabul. Docking studies indicated that conserved active site residues Met58, Thr60, Val63, Glu82, Gly84, Ser90, Asp160, Asp162, Thr169, Asn191 and Arg203 in CCoAOMT1 and CCoAOMT2 enzymes create the positive charge to balance the negatively charged caffeoyl CoA and play an important role in maintaining a functional conformation and are directly involved in donor-substrate binding.
咖啡酰辅酶A O-甲基转移酶（CCoAOMT）是一种重要的酶，参与木质素生物合成，尤其在植物细胞壁阿魏酸酯的形成过程中发挥作用。它在咖啡酰辅酶A的3-羟基甲基化过程中起关键作用。此前已从银合欢中分离出两个编码CCoAOMT的cDNA克隆，在本研究中，使用MODELLER7v7软件构建了CCoAOMT1和CCoAOMT2酶的三维模型，以找出底物结合位点。这两种蛋白质仅在两个氨基酸上存在差异，可能几乎没有或完全没有功能冗余。在溶剂化水层中进行能量最小化和分子动力学模拟后，获得了蛋白质的优化模型。通过PROCHECK、WHATCHECK、Verify_3D和ERRAT程序对模型进行了进一步评估，结果表明这些模型对于进一步的活性位点和对接分析是可靠的。优化后的模型显示，这两种蛋白质分别有9个和10个α螺旋、6个和7个β折叠。使用这些模型对接底物辅酶A（CoA）、S-腺苷甲硫氨酸（SAM）、S-腺苷高半胱氨酸（SAH）、咖啡酰辅酶A、阿魏酰辅酶A、5-羟基阿魏酰辅酶A和芥子酰辅酶A，结果表明，在有或没有SAM存在的情况下，CoA和咖啡酰辅酶A都与这些酶以高亲和力结合。因此，咖啡酰辅酶A似乎是这两种同工酶的底物。结果还表明，CoA和咖啡酰辅酶A与CCoAOMT2的结合亲和力高于CCoAOMT1。因此，CCoAOMT2的构象被认为是银合欢中存在的活性形式。对接研究表明，CCoAOMT1和CCoAOMT2酶中保守的活性位点残基甲硫氨酸58、苏氨酸60、缬氨酸63、谷氨酸82、甘氨酸84、丝氨酸90、天冬氨酸160、天冬氨酸162、苏氨酸169、天冬酰胺191和精氨酸203产生正电荷，以平衡带负电荷的咖啡酰辅酶A，在维持功能构象中起重要作用，并直接参与供体-底物结合。
